CN106841449B - It is a kind of quickly to distinguish staphylococcus aureus and the staphylococcic detection method of walsh - Google Patents
It is a kind of quickly to distinguish staphylococcus aureus and the staphylococcic detection method of walsh Download PDFInfo
- Publication number
- CN106841449B CN106841449B CN201710091672.1A CN201710091672A CN106841449B CN 106841449 B CN106841449 B CN 106841449B CN 201710091672 A CN201710091672 A CN 201710091672A CN 106841449 B CN106841449 B CN 106841449B
- Authority
- CN
- China
- Prior art keywords
- walsh
- staphylococcus aureus
- staphylococcus
- temperature
- staphylococcic
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Staphylococcus aureus and the staphylococcic detection method of walsh are quickly distinguished the invention discloses a kind of, the volatile products generated using headspace gas chromatography-mass spectrometric hyphenated technique detection staphylococcus aureus and the liquid state fermentation of walsh staphylococcus, the volatile products type generated according to two kinds of staphylococcuses is different, distinguishes staphylococcus aureus and walsh staphylococcus.The method that the present invention uses shortens 1-2 days than the existing conventional biochemical experimental identification time, can quickly distinguish staphylococcus aureus and walsh staphylococcus on plate, realizes quickly screening.The probability that a line testing staff contacts food source pathogenic bacteria is effectively reduced in this method high degree of automation, reduces the risk that food source pathogenic bacteria pollute human and environment.
Description
Technical field
Headspace gas chromatography-mass spectrometry (Headspace gas chromatography- is utilized the present invention relates to a kind of
Mass spectrometry, HS-GC-MS) technology quickly distinguishes staphylococcus aureus and the staphylococcic detection side of walsh
Method.
Background technique
Staphylococcus aureus (Staphylococcus aureus) can cause pneumonia, chordapsus etc. or even sepsis
The general infections such as disease, pyemia easily cause to poison by food；Usually enter people's by the animal foodstuff of people and animals' pyogenic infection
In vivo, being generally easy contaminated food is mainly animal food, such as beef, poultry, dairy products.By golden yellow Portugal
The food and normal diet of grape coccus pollution are not different in shape and the sense of taste, therefore are accurately and rapidly detected in food
Staphylococcus aureus has a very important significance.The detection of staphylococcus aureus mostly uses traditional biochemical reaction to test
Deng, it is long the time required to this method, 5-7 days are needed under normal circumstances, are difficult to meet the needs quickly detected.
National food safety standard " inspection of GB 4789.10-2016 food microbiological examination staphylococcus aureus "
In, staphylococcus aureus form on Baird-Parker plate: rounded, surface is smooth, colony diameter 2-3mm, face
Color is in grey black, around around with opaque circle (precipitating), often there is a clear band outside.During routine testing, walsh grape ball
Bacterium (Staphylococcus warneri) often detects in import and export food, the shape on Baird-Parker plate
State is similar to staphylococcus aureus: round, surface is smooth, moistens, colony diameter 2-3mm, and color is in grey black, around has
Precipitation ring.Therefore two kinds of staphylococcuses are difficult to differentiate between on Baird-Parker plate.Staphylococcus aureus is on blood plate
Form: bacterium colony is larger, round, and smooth bumps moisten, golden yellow (being sometimes white), the visible transparent haemolysis circle of periphery of bacterial colonies；It is fertile
Family name staphylococcus form on blood plate: bacterium colony is circle, and surface is smooth, canescence, and periphery of bacterial colonies has haemolysis circle, therefore two kinds
Staphylococcus form on blood plate is similar, is not easily distinguishable.Two kinds of staphylococcus dyeing microscopic examinations are all gram-positive cocci, row
It is spherical to arrange into grape, microscopy cannot be distinguished.
With the continuous development of modern science and technology, especially immunology, the continuous development of molecular biology, people have been created
Many quick detection methods of staphylococcus aureus are built.Round pcr is a kind of technology quick, sensitive, specificity is good,
But the technology is also to rely on the preceding increasing bacterium step of conventional method at present, often contains PCR inhibitor in enrichment liquid, thus shadow
Ring the amplification of PCR.Immunological detection method, staphylococcus aureus and walsh staphylococcus belong to staphylococcus,
Due to the presence of somatic antigen cross reaction, it is difficult to effective district parting staphylococcus aureus and walsh staphylococcus.
Summary of the invention
Staphylococcus aureus and walsh grape ball are quickly distinguished technical problem to be solved by the invention is to provide a kind of
The detection method of bacterium, to overcome the disadvantages mentioned above of the prior art, to provide the foundation and directive function of science for food safety.
Cardinal principle of the invention are as follows: utilize headspace gas chromatography-mass spectrometry (Headspace gas
Chromatography-mass spectrometry, HS-GC-MS) technology detection staphylococcus aureus and walsh grape ball
The volatile products that strains liquid fermentation generates reach differentiation according to the volatile products type difference that two kinds of staphylococcuses generate
Purpose specifically includes the following steps:
1, staphylococcus aureus and the staphylococcic culture preparation of walsh
Ring transition bacterial strain is inoculated with into the pancreas peptone soybean broth culture medium of sterilizing, 30-40 DEG C, 100-200r/min item
Under part, shaking table shaken cultivation 8-18h.It draws culture 1-10mL to be transferred in 20mL ml headspace bottle, seal, as test specimens
Product.
Wherein, pancreas peptone soybean broth component are as follows:
Tryptone 17.0g/L, phytone 3.0g/L, sodium chloride 5.0g/L, glucose 2.5g/L, dipotassium hydrogen phosphate
2.5g/L。
2, HS-GC-MS is analyzed
Head space (HS) sampling condition: 40-60 DEG C of heater box temperature, quantitative loop temperature: 85 DEG C, transmission line temperature: 100 DEG C,
Sample equilibration time: 30-60min；Chromatography column pressure: 7.7psi；Stuffing pressure: 15psi；Pressing time: 0.2min；Sample introduction is held
The continuous time: 1.0min pulls out the needle time: 0.5min.
Gas-chromatography (GC) condition: chromatographic column (0.25 μm of the μ m of 30m × 250)；Split sampling, split ratio 5:1, dottle pin
Purge flow rate is 3mL/min；Temperature program: 35-55 DEG C of holding 2-10min of initial temperature rises to 120-200 with 3-10 DEG C/min
℃；250 DEG C are risen to 5-10 DEG C/min again, keeps 1-5min, injector temperature: 250 DEG C；Carrier gas: high-pure helium (He), flow velocity:
1mL/min。
Mass spectrum (MS) condition: ion source EI, 150 DEG C of level four bars temperature, electron energy 70eV, is passed by 230 DEG C of ion source temperature
280 DEG C of defeated line temperature, acquisition mode full scan, mass scan range m/z:30-300u, solvent delay: 1-3min.
3, the qualitative and quantitative of volatile products
MS result is computed machine examination rope spectrum library and carries out qualitative analysis, while completing manual cross reference by software systems.It adopts
The peak area of volatile materials is reported with extraction ion stream mode.
Wherein, the volatile products type that two kinds of staphylococcuses generate is different, and main region divides compound to be ethyl alcohol, golden yellow
It can detecte a large amount of ethyl alcohol in the volatile products of staphylococcic liquid state fermentation, and ethyl alcohol calculated using area normalization method
Relative amount be 55%-85%, and ethyl alcohol is not detected in the volatile products of the staphylococcic liquid state fermentation of walsh, detects
To a large amount of acetic acid, and area normalization method is used to calculate the relative amount of acetic acid as 70%-81%.
Beneficial effects of the present invention:
Method of the invention shortens 1-2 days as a kind of screening implement, than the existing conventional biochemical experimental identification time, can
Quickly to distinguish the staphylococcus aureus and walsh staphylococcus on BP plate or blood plate, quickly screening is realized.Relative to
PCR method and immunization method, the screening technique the degree of automation is higher, reduces a line testing staff and contacts the several of food source pathogenic bacteria
Rate reduces the risk that food source pathogenic bacteria pollute human and environment.
Specific embodiment
The following example further illustrates the present invention, but should not make limitation of the present invention.
Fig. 1 is the volatility that walsh staphylococcus CGMCC 1.2824 and staphylococcus aureus ATCC 25923 is generated
Close the total ion chromatogram of object.
Embodiment 1
(1) prepared by the culture of staphylococcus aureus ATCC 25923 and walsh staphylococcus CGMCC 1.2824
Oese sterile working switching staphylococcus aureus ATCC 25923 arrives the sterilizing pancreas peptone soybean broth of 5mL
In, 36 DEG C, under the conditions of 150r/min, shaking table shaken cultivation 12h.It draws whole cultures and is transferred to 20mL Agilent jaw head space sample
It in product bottle, seals, as staphylococcus aureus culture to be tested.
Oese sterile working switching walsh staphylococcus CGMCC 1.2824 arrives the sterilizing pancreas peptone soybean broth of 5mL
In, 36 DEG C, under the conditions of 150r/min, shaking table shaken cultivation 12h.It draws whole cultures and is transferred to 20mL Agilent jaw head space sample
It in product bottle, seals, as walsh staphylococcus culture to be tested.
Wherein, the white peptone 17.0g/L of egg pancreas, phytone 3.0g/L, sodium chloride 5.0g/L, glucose 2.5g/L, phosphoric acid
Hydrogen dipotassium 2.5g/L.Purchased from Beijing overpass Technical responsibilities Co., Ltd.
(2) headspace gas chromatography of culture-Mass Spectrometry analysis
(it is furnished with using Agilent company of the U.S. Agilent 7890B-5977A Series gas chromatograph/mass spectrometry system
Agilent 7697A headspace autosampler) to staphylococcus aureus culture to be tested, the training to be tested of walsh staphylococcus
Support the analysis that object carries out volatile products.Headspace sampling, gas-chromatography and Mass Spectrometry Conditions are as follows:
Head space (HS) sampling condition: heater box temperature 50 C, quantitative loop temperature: 85 DEG C, transmission line temperature: 100 DEG C, sample
Equilibration time: 50min；Chromatography column pressure: 7.7psi；Stuffing pressure: 15psi；Pressing time: 0.2min；The sample introduction duration:
1.0min pulls out the needle time: 0.5min.
Gas-chromatography (GC) condition: HP-INNOWax chromatographic column (0.25 μm of the μ m of 30m × 250)；Split sampling, split ratio
For 5:1, dottle pin purge flow rate is 3mL/min；Temperature program: 50 DEG C of holding 2min of initial temperature rise to 180 DEG C with 7 DEG C/min；
250 DEG C are risen to 10 DEG C/min again, keeps 2min, injector temperature: 250 DEG C；Carrier gas: high-pure helium (He), flow velocity: 1mL/min.
Mass spectrum (MS) condition: ion source EI, 150 DEG C of level four bars temperature, electron energy 70eV, is passed by 230 DEG C of ion source temperature
280 DEG C of defeated line temperature, acquisition mode full scan, mass scan range m/z:33~300u, solvent delay: 2.2min.
(3) qualitative and quantitative of volatile products
Using the spectrum library (NIST11.L) in Agilent GCMS Chemstation software to gas chromatography-mass spectrum result into
The qualitative analysis of row compound, while manual cross reference is completed by 2.0 software systems of NIST MS Search, it is desirable that it is positive and negative
700 (matched wells) are both greater than to matching attribute (Match), possibility (Prob.%) is greater than 60.Using extraction ion stream mode
The peak area for calculating volatile materials calculates the relative amount of volatile compound using area normalization method.Choose relative amount
Representative volatile compound greater than 1% is as differentiation staphylococcus aureus and the staphylococcic classes of compounds of walsh
(table 1).
The various volatility that 1 Staphylococcus aureus ATCC 25923 and Portugal of walsh Portugal coccus CGMCC 1.2824 of table is generated
Compound
Note: CAS number is chemical substance accession number.
The volatile compound that staphylococcus aureus ATCC 25923 and walsh staphylococcus CGMCC 1.2824 is generated
Total ion chromatogram (Fig. 1) can be intuitively displayed out two kinds of staphylococcic classes of compounds difference.Wherein, walsh grape
Ethyl alcohol is not detected in the volatile compound (Fig. 1-A) of coccus CGMCC 1.2824, detects that (relative amount is a large amount of acetic acid
80.4%), detect that a large amount of ethyl alcohol are (opposite in the volatile compound of staphylococcus aureus ATCC 25923 (Fig. 1-B)
Content 65%).
Method validation
A kind of quickly to distinguish staphylococcus aureus and the staphylococcic detection method of walsh, this method passes through 10 plants of differences
The staphylococcus aureus in source and walsh staphylococcus carry out method validation, and the volatile compound of generation is shown in Table 2.The number of table 2
According to the result shows that this method can effectively distinguish the staphylococcus aureus and walsh staphylococcus of 10 plants of separate sources, method
It has good stability, by further verifying the staphylococcus aureus and walsh staphylococcus of a large amount of separate sources, can apply
Staphylococcus aureus and walsh staphylococcus are distinguished in routine testing.
The various volatile compounds that 2 staphylococcus aureus of table and walsh staphylococcus generate
Note: staphylococcus aureus ATCC29213 and staphylococcus aureus CGMCC1.1529 is reference culture；It is golden yellow
The Portugal Se Pu coccus F13-23, Staphylococcus aureus F9-17, Staphylococcus aureus F9-5, staphylococcus aureus F13-7,
Staphylococcus aureus F9-10, Staphylococcus aureus F9-12, Portugal of walsh Portugal coccus F18-2 and walsh staphylococcus F16-4
It is isolated from different food substrates, laboratory Freezing Glycerine saves.
Certainly, the above description is not a limitation of the present invention, and the present invention is also not limited to the example above, the art
Those of ordinary skill, within the essential scope of the present invention, the variations, modifications, additions or substitutions made all should belong to the present invention
Protection scope.
Claims (1)
1. a kind of quickly distinguish staphylococcus aureus and the staphylococcic detection method of walsh, which is characterized in that utilize head space
Gas chromatographyMass spectrometry detects the volatile products of staphylococcus aureus and the liquid state fermentation of walsh staphylococcus, root
The volatile products type generated according to two kinds of staphylococcuses is different, distinguishes staphylococcus aureus and walsh staphylococcus；
The detection method specifically includes the following steps:
(1) prepared by staphylococcus aureus and the staphylococcic culture of walsh
Oese transfers staphylococcus aureus and walsh staphylococcus to the pancreas peptone soybean broth culture medium to sterilize respectively
In, 30-40 DEG C, under the conditions of 120-200 r/min, shaking table shaken cultivation 8-18 h；It draws 5 mL of culture and is transferred to the top 20 mL
It in empty bottle, seals, as test sample；
Wherein, pancreas peptone soybean broth component are as follows:
17.0 g/L of tryptone, 3.0 g/L of phytone, 5.0 g/L of sodium chloride, 2.5 g/L of glucose, phosphoric acid hydrogen two
2.5 g/L of potassium；
(2) HS-GC-MS is analyzed
Head space (HS) sampling condition: 40-60 DEG C of heater box temperature, quantitative loop temperature: 85 DEG C, transmission line temperature: 100 DEG C, sample
Product equilibration time: 30-60 min；Chromatography column pressure: 7.7 psi；Stuffing pressure: 15 psi；Pressing time: 0.2 min；Sample introduction
Duration: 1.0 min are pulled out the needle time: 0.5 min；
Gas-chromatography (GC) condition: the HP-INNOWax gas chromatographic column of 30 m × 250 μm × 0.25 μm；It is diverted into
Sample, split ratio 5:1, dottle pin purge flow rate are 3 mL/min；Temperature program: 50 DEG C of holding 2min of initial temperature, with 7 DEG C/
Min rises to 180 DEG C；250 DEG C are risen to 10 DEG C/min again, keeps 2 min, injector temperature: 250 DEG C；Carrier gas: high-pure helium
(He), flow velocity: 1 mL/min；
Mass spectrum (MS) condition: ion source EI, 150 DEG C of level four bars temperature, 70 eV of electron energy, is passed by 230 DEG C of ion source temperature
280 DEG C of defeated line temperature, acquisition mode full scan, mass scan range m/z:33-300 u, solvent delay: 1-3 min；
(3) staphylococcus aureus and the staphylococcic volatile products of walsh are distinguished
Staphylococcus aureus is different from the volatile products type that walsh staphylococcus generates, and main region divides compound to be second
Alcohol can detecte a large amount of second in the volatile products that the pancreas peptone soybean broth liquid state fermentation of staphylococcus aureus generates
Alcohol, and use the relative amount of area normalization method calculating ethyl alcohol for 55%-85%, and the staphylococcic liquid state fermentation of walsh generates
Volatile products in ethyl alcohol is not detected, detect a large amount of acetic acid, and the opposite of acetic acid is calculated using area normalization method and being contained
Amount is 70%-81%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710091672.1A CN106841449B (en) | 2017-02-21 | 2017-02-21 | It is a kind of quickly to distinguish staphylococcus aureus and the staphylococcic detection method of walsh |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710091672.1A CN106841449B (en) | 2017-02-21 | 2017-02-21 | It is a kind of quickly to distinguish staphylococcus aureus and the staphylococcic detection method of walsh |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106841449A CN106841449A (en) | 2017-06-13 |
| CN106841449B true CN106841449B (en) | 2019-02-22 |
Family
ID=59134814
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710091672.1A Active CN106841449B (en) | 2017-02-21 | 2017-02-21 | It is a kind of quickly to distinguish staphylococcus aureus and the staphylococcic detection method of walsh |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106841449B (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108715886A (en) * | 2018-04-20 | 2018-10-30 | 江苏华创检测技术服务有限公司 | One kind quickly distinguishing staphylococcic method |
| CN110412168A (en) * | 2019-08-12 | 2019-11-05 | 西南民族大学 | Method based on metabolic marker analyte detection Staphylococcus aureus in food viable bacteria |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104483409B (en) * | 2014-12-12 | 2017-01-11 | 河北农业大学 | Fingerprint based staphylococcus aureus identification method |
-
2017
- 2017-02-21 CN CN201710091672.1A patent/CN106841449B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN106841449A (en) | 2017-06-13 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Lambert et al. | Cellular fatty acid compositions and isoprenoid quinone contents of 23 Legionella species | |
| Ahamed et al. | Production of hydrocarbon compounds by endophytic fungi Gliocladium species grown on cellulose | |
| US20220003763A1 (en) | Inert Carrier Salmonella and Potential Use Thereof | |
| CN106841449B (en) | It is a kind of quickly to distinguish staphylococcus aureus and the staphylococcic detection method of walsh | |
| Wang et al. | Rapid identification of Staphylococcus aureus, Vibrio parahaemolyticus and Shigella sonnei in foods by solid phase microextraction coupled with gas chromatography–mass spectrometry | |
| Qiu et al. | High-temperature induced changes of extracellular metabolites in Pleurotus ostreatus and their positive effects on the growth of Trichoderma asperellum | |
| Peng et al. | Comparative analysis of astaxanthin and its esters in the mutant E1 of Haematococcus pluvialis and other green algae by HPLC with a C30 column | |
| CN104531885A (en) | Aeromonas veronii rapid detection primer, kit and application | |
| CN111972645B (en) | Method for producing pickle | |
| CN107064335B (en) | A kind of detection method of quick differentiation salmonella typhimurium and proteus mirabilis | |
| CN108693004A (en) | A kind of method that antibiotic filter residue whether is adulterated in detection rapeseed dregs | |
| Alley et al. | Electron capture gas-liquid chromatographic-mass spectral identification of acids produced by Neisseria meningitidis in a defined medium | |
| Alvarez-Rivera et al. | A novel outlook on detecting microbial contamination in cosmetic products: analysis of biomarker volatile compounds by solid-phase microextraction gas chromatography-mass spectrometry | |
| CN101570781B (en) | Detection kit and species-based detection method for lactobacilli | |
| Lewis et al. | Determination of volatile acid production of Clostridium by gas chromatography | |
| CN106442795B (en) | A kind of detection method of quick differentiation Listeria monocytogenes and Si Shi Listeria | |
| CN103981270B (en) | Mermaid luminous bacillus rapid detection primer, test kit and application | |
| CN101285048B (en) | Rhodobacter sphaeroides mutant strain and uses thereof | |
| CN107271490B (en) | The method that Antrodia camphorata liquid fermentation process quickly characterizes triterpenoid changes of contents | |
| CN111925962B (en) | Pediococcus ethanolica capable of producing mannitol and diallyl disulfide | |
| CN101936960B (en) | Analytical method of components of fatty acid contained in listeria cells | |
| Larsson et al. | Characterization of Clostridium difficile and its differentiation from Clostridium sporogenes by automatic head-space gas chromatography | |
| Jackson et al. | Fatty acids of a non-pigmented, thermophilic bacterium similar to Thermus aquaticus | |
| Rahaman et al. | Isolation, identification and characterization of Clostridium perfringens from lamb dysentery in Dinajpur district of Bangladesh | |
| CN105624299A (en) | High-pathogenicity Vibrio parahaemolyticus rapid detection primer and kit |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP02 | Change in the address of a patent holder | ||
| CP02 | Change in the address of a patent holder |
Address after: 266000 No. 83, Xinyue Road, Chengyang District, Qingdao City, Shandong Province Patentee after: SHANDONG TECHNICAL CENTER OF INSPECTION AND QUARANTINE Address before: 266002 Shandong province Qingdao city qutangxia Road No. 70 Patentee before: SHANDONG TECHNICAL CENTER OF INSPECTION AND QUARANTINE |